Xia, J.; Yan, J.; Tang, M.; Zhao, B.; Chen, K.

Multiplexed fluorescence imaging is limited by spectral overlap and the small number of excitation or emission channels available on standard microscopes, restricting most laboratories to low-plex imaging. Here we introduce physics-informed spectra-free multiplexed imaging (PhySMI), a self-supervised framework for underdetermined spectral unmixing that enables highly multiplexed imaging without dense spectral measurements after training. By embedding the spectral forward-mixing process into a self-consistent architecture, PhySMI recovers physically plausible source decompositions from unlabeled data without paired ground-truth labels while suppressing stochastic acquisition noise. PhySMI resolves five subcellular structures from only three excitation channels, overcoming the conventional channel-number limit while preserving spectral fidelity and minimizing crosstalk (<0.5%). The framework also generalizes across imaging systems, enabling zero-shot deployment on standard fluorescence microscopes. In live cells, PhySMI enables fast five-color imaging of dynamic multi-organelle interactions with improved temporal resolution and reduced photobleaching and phototoxicity relative to conventional spectral imaging. These results establish a general strategy for physics-informed learning in underdetermined imaging inverse problems and represent a step toward a general-purpose framework for highly multiplexed fluorescence imaging on standard microscopy platforms.